Austin Fife1, Daniel Carrillo2, Gary Knox3, Fanny Iriarte4, Kishore Dey5, Avijit Roy6, Ronald Ochoa7, Gary Bauchan8 \(\dagger\), Mathews Paret4,9, Xavier Martini1*
1 University of Florida, Department of Entomology and Nematology, North Florida Research and Education Center, Quincy FL 32351
2 University of Florida, Department of Entomology and Nematology, Tropical Research and Education Center, Homestead FL 33031
3 University of Florida, Department of Environmental Horticulture, North Florida Research and Education Center, Quincy FL 32351
4 University of Florida, Department of Plant Pathology, North Florida Research and Education Center, Quincy FL 32351
5 The Florida Department of Agriculture and Consumer Services, Division of Plant Industry, Section of Plant Pathology, Doyle Conner Building, 1911 SW 34th street, Gainesville, FL 32608
6 United States Department of Agriculture – Agriculture Research Service, Molecular Plant Pathology Laboratory, 10300 Baltimore Ave, Bldg. 4 BARC-West, Beltsville, MD 20705
7 United States Department of Agriculture - Agriculture Research Service, Systematic Entomology Laboratory 10300 Baltimore Ave, Bldg. 5 BARC-West, Beltsville, MD 20705
8 United States Department of Agriculture - Animal and Plant Health Inspection Service, Electron and Confocal Microscopy Unit, Bldg. 12 BARC-West, 10300 Baltimore Ave, Beltsville, MD 20705
9 Plant Pathology Department, University of Florida, Gainesville, FL 32611
*Corresponding author; E-mail: xmartini@ufl.edu Phone: 850-875-7160 Fax: 352-846-6617
Orchid Fleck Virus (OFV) is the type species for the genus Dichorhavirus, a plant pathogen which infects more than fifty plant species belonging to the family Orchidaceae, Asparagaceae (Nolinoidaea), and Rutaceae (Citrus). The sole vectors for dichorhaviruses are mites from the genus Brevipalpus Donnadieu (Trombidiformes: Tenuipalpidae), which are also known to spread cileviruses. OFV was found infecting liriopogons Liriope spp. & Ophiopogon spp. (Asparagaceae: Nolinoidaea) and Aspidistra elatior Blume (Asparagaceae: Nolinoidaea), during the summer of 2020 in a landscape in Leon County, Florida. OFV specific conventional reverse transcription polymerase chain assay (RT-PCR) assay and Sanger sequencing. RT-PCR amplicons had a 98% identity with the known OFV sequences available in the GenBank (Accession numbers: AB244418 and LC222630). Virus identity was also confirmed via quantitative RT-PCR (RT-qPCR). Additional leaf samples were collected from other putatively infected Asparagaceae from Leon and Alachua counties. Identification of partial genome sequence confirmed the presence of both OFV orchid strains (OFV-Orc1 and OFV-Orc2) in Florida. These strains of OFV are known to infect citrus and cause citrus leprosis disease. Three potential vector species were identified from OFV-infected plants: Brevipalpus californicus sensu lato, B. obovatus Donnadieu and B. confusus Banks. Florida has various Brevipalpus susceptible native and introduced plant species in the landscape. In this study we report three new hosts from the family Asperagaceae from multiple locations. Our data suggested that the OFV widely distributed in Florida and might be a threat for various plant species and possibly citrus growing in the southeastern US.
False spider mite, flat mite, Brevipalpus-transmitted viruses, Liriope, Nolinoideae, Ophiopogon, Ruscaceae, Rutaceae, Asparagaceae, orchid, Orchidaceae, pests, ornamental plants, orchid fleck virus.
Orchid fleck dichorhavirus (OFV) is the type member for the genus Dichorhavirus, family Rhabdoviridae; a bacilliform, nuclear rhabdovirus composed of two segments of single-stranded, negative-sense RNA which infects plants (Dietzgen et al. 2014, Walker et al. 2018, Amarasinghe et al. 2019). Flat mites from the genus Brevipalpus Donnadieu (Trombidiformes: Tenuipalpidae) are the sole vectors for cileviruses and dichorhaviruses (Maeda 1998), and B. californicus (Banks) group of mites are the only known to transmit OFV in a persistent propagative manner (Kondo et al. 2003).
OFV-infected plants exhibit various symptoms depending on the infected plant species as well as the strain of the OFV associated with the infection (Kubo et al. 2009), but symptoms typically appear as chlorotic flecks, which ultimately coalesce into larger spots or ringspot patterns (Fig. 1, Fig. 2).
OFV was first described infecting Cymbidium orchids in Japan (Doi et al. 1977). OFV and OFV-like rhabdoviruses have been reported infecting orchids in Asia, Africa, North America, South America, Europe, and Oceania. The prevalence of OFV and its mite vector is thought to be associated with the movement of infected orchids (Dietzgen, Freitas-Astúa, et al. 2018).
OFV naturally infects more than fifty species of Orchidaceae (Kitajima et al. 2010, Peng et al. 2013), some Asparagaceae (Nolinoidaea) (Mei et al. 2016, Dietzgen, Tassi, et al. 2018), and Rutaceae: (Citrus), where it causes citrus leprosis-like symptoms (Roy et al. 2015, 2020, Cook et al. 2019, Olmedo-Velarde et al. 2019). Mechanical transmission of OFV is possible to some plants belonging to the plant families Chenopodiaceae, Aizoaceae, Fabaceae, and Solanaceae (Chang et al. 1976, Kondo et al. 2003, Peng et al. 2013), under laboratory conditions.
During June 2020, chlorotic ringspot symptoms were observed on Giant Lilyturf Liriope spp., cv. ‘Gigantea’ in a landscape of Leon County, Florida (Fig. 1). Liriope belong to a group of plants in the family Asparagaceae, subfamily Nolinoidaea, which includes a diverse array of various monocotyledonous southeastern Asia native liliod plants (Chase et al. 2009, Meng et al. 2021). Liriope and the closely related Ophiopogon Ker Gawler (Asparagaceae: Nolinoidaea) are considered the most important ground cover nursery plants in the southeastern United States (Mcharo et al. 2003).
Viral infections of suspected leaf samples were initially tested at the Plant Disease Diagnostic Clinic at the North Florida Research and Education Center (NFREC) in Quincy, FL. All the samples were tested with RT-PCR and were found negative for begomovirus, carlavirus, potyvirus, tospovirus as well as for Cucumber mosaic virus, Impatiens necrotic spot virus, Tobacco mosaic virus and Tomato spotted wilt virus.
The initial site of collection was visited two more times during 2020 to gather plants for identification of the unidentified plant pathogen. These surveys were conducted during July and August to collect more putatively infected plants, including more Liriope spp. as well as a new species which also belongs to the family Asparagaceae; Aspidistra elatior Blume, which was suspected to be infected due to unusually chlorotic leaves (Fig. 2). Upon collection, these new samples were sent to the Florida Department of Agriculture and Consumer Services (FDACS) for identification.
The presence of OFV was confirmed using OFV generic R2-Dicho-GF and R2-Dicho-GR primers by one step conventional RT-PCR (Roy et al. 2020), amplifying ~800 nt of L-gene (RNA2) amplicon from an infected Liriope leaf sample. Sanger sequencing of RT-PCR amplicons shared 98% nucleotide identity with orchid strains of OFV: OFV-Orc1 and OFV-Orc2 (GenBank Accession numbers: AB244418 and LC222630) (Kondo et al. 2006, 2017).
Subsequent surveys of plants belonging to the subfamily Nolinoidaea in Florida have revealed more sites with Asparagaceae putatively infected with OFV in Leon and Alachua counties (Table 1). Mites were collected from symptomatic plants in Leon county and observed with phase contrast microscopy. Tenuipalpid mites (flat mites or false spider mites) were commonly found in abundance on the Asparagaceae which tested positive for OFV. These flat mites were originally identified as Brevipalpus californicus (Banks) sensu lato and later confirmed by the FDACS via Differential Interference Contrast (DIC) microscopy. The Brevipalpus mite species complex is known to contain cryptic species (Childers and Rodrigues 2011) that require advanced microscopy techniques, such as cryo scanning electron microscopy (Cryo-SEM) for species identification (León and Nadler 2010, Beard et al. 2015, Skoracka et al. 2015). Additional mite samples were collected from the original OFV detection site, and examined under Cryo-SEM (Fig. 3). The determinations approved prior identifications of B. californicus s.l. but revealed the presence of two other species B. obovatus and B. confusus (Fig. 4).
The first report of OFV in the US is thought to be Ko et al. (1985), who describes nuclear inclusions caused by an undescribed bacilliform rhabdovirus in Brassia orchids. The significance of this report is their reference to spoke-wheel configurations of the viral particles (Ko et al. 1985), a sign typically associated with OFV infection (Chang et al. 1976). Unfortunately, Ko et al. (1985) made no mention of mites or further investigations of this virus. The first certain report of OFV was from Hawaii in 2001 (Blanchfield et al. 2001), while the first report from the continental US was by Bratsch et al. (2015). In that publication, the authors confirmed the presence of OFV in Phalaenopsis hybrids in the US, using TEM of ultrathin sections of plant tissue as well as molecular sequence analysis and its association with Brevipalpus mites (Bratsch et al. 2015). The authors did not make a conclusive species identification but suggested the mite vector was within the B. californicus group, as referred by Kondo et al. (2003).
OFV has been reported in other Nolinoidaea in Australia (Mei et al. 2016, Dietzgen, Tassi, et al. 2018), including Liriope spicata (Thunb.) Lour. (Mei et al. 2016) but not in the US. The Florida collected plants of Liriope spp., cv ‘Gigantea’ are thought to belong to either Liriope muscari or Liriope gigantea. We are not aware of any previous report of OFV infection in Ophiopogon plants. Although Zheng et al. (2013) mentions the association of B. californicus with A. elatior but they did not mention the presence of OFV symptoms in this plant. However, our finding will be notified as the first report of OFV in the US on ornamentals and among them A. elatior is a new natural host of OFV.
The dichorhavirus that infects citrus in Hawaii, Mexico, Colombia, and South Africa are identical to the OFV in gene order, content, and the genome sequence. According to the International committee on Taxonomy of Viruses (ICTV) classification, OFV consist of two orchid strains (OFV-Orc1 and OFV-Orc2) and two citrus strains (OFV-Cit1 and OFV-Cit2). Both the orchid strains of OFV infects citrus (Roy et al. 2020), but none of the citrus strains have been reported from any orchid species. Detecting OFV in Florida represents a concern for horticulturists who grow orchids, Liriope, Ophiopogon, or other susceptible Asparagaceae species which are commonly used in landscaping. Florida is also home to a plethora of native and naturalized orchid species, many of which are threatened, including cultivating Vanilla in southern Florida (Chambers et al. 2019) and the famous Ghost Orchid, [Dendrophylax lindenii (Lindl.) Benth. ex Rolfe]. Citrus leprosis was present in Florida during the 1860’s and eradicated in the mid-1960s. In fact, Kitajima et al. (2011) found that the Citrus Leprosis virus (CiLV) which previously affected Florida citrus was a nuclear type of citrus leprosis closely related to OFV strains (Hartung et al. 2015, Roy et al. 2020). Association of a distant relative of OFV named Citrus leprosis dichorhavirus-N0 (CiLV-N0) was confirmed in relation to the leprosis disease outbreak in Florida (Hartung et al. 2015). The recent detection of OFV-Orc1 in South Africa (Cook et al. 2019) in C. sinensis (Navel and Valencia orange) and OFV-Orc2 in Hawaii (Velarde et al. 2021) in C. reticulata (mandarin) and C. jambhiri (rough lemon) associated with leprosis-like symptoms highlights the threat of different strains of OFV on citrus; which will be a definite concern to the multi-billion dollar citrus industry. B. californicus and B. yothersi are both known vectors of Dichorhaviruses (OFV) and Cileviruses (Citrus Leprosis) (Knorr 1968, Kondo et al. 2003, Beltran-Beltran et al. 2020) and B. obovatus is a suspected vector as well (Childers et al. 2003). All three mite species/complexes are present in Florida (Childers et al. 2003, Akyazi et al. 2017) (Fig. 4). Therefore, it is critical to identify the vector of OFVs in Florida and monitor its spread to determine the risk this virus represents for the native plants, agriculture and the ornamental/landscaping industries of Florida and the surrounding regions.
We would like to give a special thanks to the Tallahassee Museum for their patience, cooperation, and support with collecting plant samples. We also want to thank Drs. Sam Bolton, FDACS and Aline Tassi, Univ. of Sao Paulo, Brazil for checking the mites we have sent for species validation. Furthermore we are grateful for Dr. Marc S. Frank’s identification of the Liriopogons collected. We are especially indebted to the late Dr. Gary Bauchan for his contributions to this study and the field of acarology, he will be greatly missed. This research was partly funded by the USDA National Institute of Food and Agriculture, Hatch project FLA-NFC-005607. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA; USDA is an equal opportunity provider and employer.
| Scientific Name | Common Names | Symptoms Observed | County |
|---|---|---|---|
| Liriope muscari Bailey | Lilyturf, Orchardgrass, Monkeygrass | Ringspots, Chlorotic Flecking, Necrotic Lesions | Alachua & Leon |
| Liriope gigantea* Hume | Giant Lilyturf | Ringspots, Chlorotic Flecking, Necrotic Lesions | Alachua & Leon |
| Ophiopogon japonicus Ker Gawl. | Dwarf Lilyturf, Mondo Grass, Snake’s beard | Ringspots, Chlorotic Flecking, Necrotic Lesions | Leon |
| Ophiopogon intermedius** Don | Aztec Grass, ‘Argenteomarginatus’ | Ringspots, Chlorotic Flecking, Necrotic Lesions | Alachua & Leon |
| Aspidistra elatior Blume | Cast Iron Plant, Bar-room Plant | Chlorosis, Chlorotic Flecking, Necrotic Lesions | Leon |
Table 1: List of plants with symptoms of Orchid fleck dichorhavirus found in northern Florida. * L. gigantea have been traditionally classified as seperate from L. muscari by Broussard (2007) and Fantz et al. (2015), although this distinction has been challenged by Wang et al. (2014) and Masiero et al. (2020). * * O. intermedius is sometimes misclassified as Liriope muscari ‘Variegated Evergreen Giant’ Fantz (2009) or ‘Grandiflora White’ (Fantz 2009).
Fig. 1: Variety of symptoms expressed by Liriope spp. infected with Orchid fleck dichorhavirus (OFV): (a) ringspot symptoms on Liriope gigantea (b-c) Details of ringspot symptoms on Liriope gigantea (d) chlorotic ringspots Liriope muscari cv. ‘Silvery Sunproof’
Fig. 2: Symptoms expressed by Aspidistra elatior infected with Orchid fleck dichorhavirus (OFV): (a) Detail of leaf chlorosis (b) Chlorosis caused by OFV appears similar to sunburn damage (c-d) Chlorotic ringspots may indicate early symptoms of OFV
Fig. 3: Cryo-SEM images of Brevipalpus californicus sensu lato displaying various characters used for identification (Baker and Tuttle 1987, Beard et al. 2012) (a) Dorsum (b) Lateral view (c) Venter (d) Close up of distal end of leg 2, with arrows indicating paired solenidia, characteristic of the genus Brevipalpus (e) Enlargement of the microplates of the mite cerotegument (f) Dorsal view of the distal portion of mite abdomen (g) Dorsal view of the mite rostrum (h) Ventral view of mite rostrum, observe 3 distal setae.
Fig. 4: Florida is home to other common pest species of Brevipalpus which are potential vectors of Orchid fleck dichorhavirus: (a) B. yothersi, dorsal (b) B. yothersi, lateral (c) B. obovatus, dorsal.